Hindawi Publishing Corporation International Journal of Food Science Volume 2013, Article ID 585931, 12 pages http://dx.doi.org/10.1155/2013/585931

Review Article Ethoxyquin: An Used in Animal Feed

Alina BBaszczyk,1 Aleksandra Augustyniak,1 and Janusz Skolimowski2

1 Department of General Genetics, Molecular Biology and Plant Biotechnology, Faculty of Biology and Environmental Protection, University of Łod´ z,Banacha12/16,90-237Ł´ od´ z,´ Poland 2 Department of Organic Chemistry, Faculty of Chemistry, University of Łod´ z,´ Tamka 12, 91-403 Łod´ z,´ Poland

Correspondence should be addressed to Alina Błaszczyk; [email protected]

Received 15 January 2013; Accepted 2 April 2013

Academic Editor: Angel´ Medina-Vaya´

Copyright © 2013 Alina Błaszczyk et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ethoxyquin (EQ, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) is widely used in animal feed in order to protect it against lipid peroxidation. EQ cannot be used in any food for human consumption (except spices, e.g., chili), but it can pass from feed to farmed fish, poultry, and eggs, so human beings can be exposed to this antioxidant. The manufacturer Monsanto Company (USA) performed a series of tests on ethoxyquin which showed its safety. Nevertheless, some harmful effects in animals and people occupationally exposed to it were observed in 1980’s which resulted in the new studies undertaken to reevaluate its toxicity. Here, we present the characteristics of the compound and results of the research, concerning, for example, products of its metabolism and oxidation or searching for new on the EQ backbone.

1. Introduction pharmaceuticals and cosmetics. There are many controversies abouttheuseofthesetwoantioxidantsinfoods.Some During storage of animal feed many different processes may experimental studies have reported that both BHT and occur which alter their initial natural proprieties. First of all, BHA have tumour-promoting activity [1, 2]. On the other lipids undergo peroxidation, the process during which they hand, there were reports on anticarcinogenic properties of are deteriorated in a free radical autocatalytic oxidation chain these antioxidants when they are used at low concentrations reaction with atmospheric oxygen. Lipid autooxidation is a [3]. Human exposures are at least 1000-fold below those cascade phenomenon ensuring continuous delivery of free associated with any neoplastic actions in laboratory animals radicals, which initiate continuous peroxidation. This process thusitisassumedthattheyarenotharmfulforhumanbeings results in food rancidity which manifests itself as the change [3, 4]. of taste, scent, and color as well as decrease in shelf life of the The third compound, EQ, is one of the best known feed product. Natural or synthetic antioxidants are usually used to antioxidants for domestic animal and fish. Its unquestionable slow down or stop lipid peroxidation and in consequence to advantage is its high antioxidant capacity and low production preserve freshness of the product. Many natural antioxidants, costs. However, some of the authors have suggested that it such as tocopherols, vitamin C, flavonoids, for a short is responsible for a wide range of health-related problems period, may be effective in food preserving, but in many in dogs as well as in humans [5–9]. Due to the increased cases such protection is not sufficient. Therefore synthetic use of this antioxidant it was nominated by FDA (US Food antioxidants are widely used, among which BHT (butylated and Drug Administration) for carcinogenicity testing [10]. hydroxytoluene), BHA (butylated hydroxyanisole), and EQ ThetestswerecarriedoutbyMonsantoCompany(USA),EQ (ethoxyquin) are the most frequent. However, some effects of producer, and after that in 1977 FDA requested for optional synthetic antioxidants are not always beneficial for our health. lowering of the maximum level of EQ in complete dog foods Antioxidants such as BHA or BHT have been widely used from allowed 150 ppm (0.015%) to 75 ppm (0.0075%). At the for many years to preserve freshness, flavor, and colour of same time new studies were started by the Pet Food Institute foodsandanimalfeedsaswellastoimprovethestabilityof to determine whether even lower EQ levels (between 30 and 2 International Journal of Food Science

60 ppm) would provide antioxidant protection for dog food Table 1: Permitted amounts of EQ in different products approved ∗ [11]. by FDA . Ethoxyquin is also known as Santoquin, Santoflex, Product Dose [ppm] Quinol. It was originally developed in rubber industry to prevent rubber from cracking due to oxidation of isoprene Animal feed 150 [12]. The Monsanto Company (USA) taking into account its Spices 150 high antioxidant efficiency and stability as well as low costs Uncooked fat of meat from animals (except poultry) 5 of synthesis refined it later for use as a preservative in animal Uncookedliverandfatofpoultry 3 feeds because it protects against lipid peroxidation and Uncooked muscle meat of animals 0.5 stabilizes fat soluble vitamins (A, E). Presently, ethoxyquin is Poultry eggs 0.5 used primarily as an antioxidant in canned pet food and in Milk 0 feed intended for farmed fish or poultry. Pear 3 Theuseofethoxyquinisnotpermittedinfoodsintended ∗ According to the Code of Federal Regulations (CFR), Title 21, Parts 573.380, for human, except preserving powdered paprika and chili 573.400, 172.140. colour and using it as an antiscald agent in pears and apples (inhibition of “brown spots” development). However, because EQ is used as a feed antioxidant it can be also found in other products intended for human consumption like fish meal, fish The other metabolic pathways include hydroxylation and oils, and other oils, fats, and meat (Table 1). An acceptable glucuronidation at position 8-C, deethylation at 6-C and −1 daily intake (ADI) of EQ for human (0–0.005 mg kg bw) epoxidation between positions 3-C and 4-C [21]. The main based on the results obtained from studies on dogs was metabolites observed may be different depending on ani- established in 1998 [13, 14]. mal species. In mice mainly glucuronide metabolites were This paper presents characteristics of ethoxyquin with detected while in rats those result from conjugation of EQ regard to its properties, metabolism, toxicity, possible car- with sulfate. cinogenicity, and antioxidant activity. In the studies of Bohne et al. [23, 26] parent EQ, dem- ethylated EQ (DEQ), quinone imine (QI), and EQ dimer (EQDM) were observed in salmonid fish after long-term 2. Physical and Chemical Properties of dietary exposure to EQ. It was in agreement with the results Ethoxyquin (EQ) obtained earlier by Skaare and Roald [27]. EQ is considered asamodelinducerofphaseIIenzymesinvolvedinthe For the first time EQ was synthesized in 1921 by Knoevenagel metabolism of xenobiotics, but influence of EQ on phase I [15]. The synthesis was based on condensation of aniline with enzyme gene transcript levels was also observed [28, 29]. The molecules of acetone or its analogues [15, 16]. Błaszczyk et keyroleinmediatingphaseIreactions(e.g.,oxidationor al. [16] synthesized EQ from p-phenetidine (4-ethoxyaniline) reduction) producing more hydrophilic compounds is played and diacetone alcohol in the presence of p-toluenesulfonic by the CYP (cytochrome P450) enzyme family. Bohne et al. acid or iodine. Pure ethoxyquin (EQ; 6-ethoxy-1,2-dihydro- [28, 29] observed the alteration of CYP3A gene expression; 2,2,4-trimethylquinoline; CAS number 91-53-2; Figure 1)is an increase in the amount of CYP3A transcripts was detected a light yellow liquid, but it changes color to brown if it in salmon after feeding them with the diet containing EQ at −1 is exposed to oxygen [12]. It also tends to polymerize on the highest dose used (1800 mg kg ). The authors speculate exposure to light and air. The scent of EQ is described as that EQ may regulate CYP3 gene expression by interaction, mercaptan like. As a nonpolar substance EQ is soluble only for example, with pregnane X nuclear receptor (PXR) whose in organic solvents. Some of the additional properties of EQ function is to sense the presence of toxic xenobiotics and are presented in Table 2. in response enhance the expression of proteins involved in their detoxification. On the other hand, CYP1A1 gene 3. Biotransformation of EQ expression, which was described as an exposure biomarker to both endogenous and exogenous compounds [30], was 3.1. Animals. Ethoxyquin is rapidly absorbed from gastroin- not increased after dietary exposure of salmonid fish toEQ testinal tract of laboratory animals like rats and mice. Peak and during the depuration period a trend toward down- blood concentration of the compound is observed within regulation was noted [28, 29]. Such an effect was observed 1h.DistributionofEQinanimalbodyissimilarwhenit despite the increase in the expression of AhR mRNA (AhR, is administered orally and intravenously. Small amounts of cytosolic transcription factor responsible for changes in gene parent EQ were detected in liver, kidney, and adipose tissue transcription). This effect can be explained in several ways. and fish muscles [20–24]. It is excreted predominantly as For example, the parent EQ may bind CYP1A1 protein metabolites via urine. Metabolism of EQ was studied in rats, and as a result may inhibit the gene expression and activ- mice, dogs, chickens, and fish, as well as in plants [13, 21, 25]. ity of protein [31]. Hepatic antioxidant response elements It is not fully described but some metabolites were identified (ARE) or AhR repressor (AhRR) together with basic-helix- (Table 3). The most important EQ metabolites observed in loop-helix-PAS (Per-AhR/ARNT-Sim homology sequence) raturineandbileresultfromO-deethylationatposition6-C, of transcription factor usually associated with each other to and then conjugation with sulphate or glucuronide residues. form heterodimers (AhR/ARNT or AhRR/ARNT) may be International Journal of Food Science 3

Table 2: Physical and chemical properties of ethoxyquin.

Properties of EQ

Molecular formula C14H19NO −1 Molecularmass 217.34[gmol ] IUPAC name 1,2-dihydro-2,2,4-trimethylquinolin-6-yl ethyl ether CAS name 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline Chemical class ∘ Melting point 0 Ca ∘ Boiling point 123–125 Cat2mmHga Insoluble in water, soluble in fat and animal and plant oils, and soluble in ethanol, methanol, DMSO, and Solubility DMF Vapor density 7.48 (AIR = 1)a ∘ Stability Stable under ordinary conditions, storage temp. 0–6 C 𝑛 ∘ a Spectral properties Index of refraction: 𝐷 1.569–1.672 at 25 C Max absorption: 354 nm. Intense mass spectral peaks: 202 m/z (100%), 108 m/z (53%), 174 m/z (48%), and 137 m/z (36%)a 13 𝛿 CNMRinCDCl3 EQ (8% solution) 𝐶 in ppm: 151.2 (EQ-C-6); 137.6 (EQ-C-4); 129.5 (EQ-C-3); 128.5 (EQ-C-9); 122.8 (EQ-C-10); 114.6 (EQ-C-8); 113.6 (EQ-C-7); 111.1 (EQ-C-5); 64.2 (EQ-CH2-O); 51.6 b (EQ-C-2); 30.4 (EQ-CH3-4); 18.5 (EQ-CH3-4); 15.0 (EQ-CH3-CH2-O-) aAccording to the National Library of Medicine HSDB Database (last revision date: 2003) [18]. bAccording to Błaszczyk and Skolimowski [19]. DMSO: dimethyl sulfoxide, DMF: N,N-dimethylformamide. also involved in the CYP1A1 downregulation process. These shown that in general they are different from those observed heterodimers can influence gene expression by binding ARE in animals (Table 3). In pears treated with ring-labeled 14 sequences in the gene promoter regions [32]. [ C]ethoxyquin the following compounds were detected: N– However, EQ as other phenolic antioxidants, first of N and C–N dimers, demethylethoxyquin (DMEQ), dehy- all causes induction of phase II xenobiotic-metabolizing drodemethylethoxyquin (DHMEQ), and dihydroethoxyquin enzymes. Bohne et al. [28, 29] observed elevated dose-related (DHEQ) [14, 25]. It was shown that ethoxyquin was rapidly uridine diphosphate glucuronosyl-transferase (UDPGT) degradated or metabolized but itself it was not translocated mRNA expression after dietary exposure to EQ. As UDPGT into the pulp of fruit where the residues were detected (less reacts with the compounds that have the hydroxyl group than 0.5% of total radioactive residue was EQ). Toxicity of (-OH) parent EQ cannot be the potential substrate for EQ metabolites, MEQ, DHMEQ, and DHEQ was studied in −1 glucuronidation, only its metabolites, for example DEQ dogs (oral administration, single doses of 50 to 200 mg kg (6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline; Table 3), bw),anditwasfoundthattheydidnotshowanysignificant the metabolite identified by Berdikova Bohne et al.26 [ ]in toxicity. In the report of Gupta and Boobis [14]therank Atlantic salmon. Changes in the expression of glutathione order of toxic potency for the plant metabolites and EQ is S-transferase (GST) gene were also observed after feeding MEQ < EQ < DHEQ < DHMEQ (the least toxic first). MEQ, animals with EQ containing feed. The alterations in GST DHMEQ, and DHEQ were also evaluated for genotoxicity in activity caused by EQ were documented in Atlantic salmon in vitro and in vivo tests. The compounds did not cause gene [28, 29], in rodents [33, 34], and in nonhuman primates mutations in Salmonella typhimurium and Escherichia coli [35]. In addition to UDPGT and GST, some other enzymes strains, but they induced chromosomal aberrations or/and are involved in phase II metabolism of EQ, for example, endoreduplication in Chinese hamster ovary cells. On the NADP(H) : quinone oxidoreductase and epoxide hydrolase other hand, plant metabolites/degradation products did not [36]. exhibit genotoxic potential in vivo. ADI intake for humans for The expression pattern of both phase I and II enzymes MEQ, DHMEQ, and DHEQ was estimated at the same level −1 involved in EQ metabolism may vary in different animals and as for EQ (0–0.005 mg kg bw). should be considered in relation to the ratio of parent EQ and its metabolites (first of all DEQ, QI, and EQDM) in the liver [29, 37]. The research concerning this issue is currently 4. Antioxidant Activity of EQ in progress. EQ possesses high-antioxidative activity. It is very efficient in protecting lipids which are present in food against oxidization 3.2. Plants. Ethoxyquin is also registered as an antioxidant to [38, 39]. Specifically it is used to retard oxidation of carotene, control scald (browning) in apples and pears. The EQ plant xanthophylls, and vitamins (like vitamins A or E). In animals metabolites/degradation products were detected, and it was treatedwithethoxyquinthreetimeshigherlevelofvitamins 4 International Journal of Food Science

Table 3: Metabolite/degradation products of EQ detected in different organisms.

EQ metabolite/degradation product Organism OH CH3 O S O O 1,2-Dihydro-6-hydroxy-2,2,4-trimethylquinoline sulphate Rats1 CH3 N H CH3 CH3 Glu O

1 CH3 1,2-Dihydro-6-hydroxy-2,2,4-trimethylquinoline glucuronide Mice N H CH3

OH CH2 O S O OH O 1,2,3,4-Tetrahydro-3,6-dihydroxy-4-metylene-2,2-dimethylquinoline sulphate Rats1 CH3 N H CH3

CH3 Et O Rats1 CH 1,2-Dihydro-6-ethoxy-8-hydroxy-2,2,4-trimethylquinoline glucuronide 3 Mice1 N Glu O H CH3

CH3 O Rats1 8-(S-glutathionyl)-2,2,4-trimethylquinol-6-one CH3 Mice1 G S N CH3

H3C S G Et O OH 1,2,3,4-Tetrahydro-6-ethoxy-3-hydroxy-4-(S-glutathionyl)-2,2,4- Rats1 CH3 trimethylquinoline Mice1 N H CH3

CH3 O Rats1 CH 2,6-Dihydro-2,2,4-trimethyl-6-quinolone (quinone imine (QI)) 3 Atlantic salmon2 N CH3

CH3 HO

6-Hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline (deethylated EQ (DEQ)) Atlantic salmon2 CH3 N H CH3

CH3 Et O 󸀠 CH 1,8 -Di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) or 3 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 N 6,6 -diethoxy-2,2,2 ,2 ,4,4 -hexamethyl-1 ,2 -dihydro-2H-1,8 -biquinoline CH3 2 ∗ (ethoxyquin dimer (EQDM)) Atlantic salmon H C H ∗ 3 N Instead of the hydrogen atom there is a methyl group, it gives methyl C-N Pears3,4 H3C ethoxyquin dimer, 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 6,6 -Diethoxy-1 ,2,2,2 ,2 ,4,4 -heptamethyl-1 ,2 -dihydro-2H-1,8 -biquinoline O Et

H3C International Journal of Food Science 5

Table 3: Continued. EQ metabolite/degradation product Organism CH3 Et O

CH3 N CH3 N-N Ethoxyquin dimer, 󸀠 1,1 -di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) or Pears3,4 CH3 󸀠 󸀠 󸀠 󸀠 󸀠 󸀠 N 6,6 -diethoxy-2,2,2 ,2 ,4,4 -hexamethyl-2H,2 H-1,1 -biquinoline CH3 Et O CH3

CH3 Et O

6-Ethoxy-1,2,2,4-tetramethyl-1,2-dihydroquinoline, CH3 Pears3,4 N methylethoxyquin (MEQ) CH3 CH3

CH3 Et O 6-Ethoxy-2,4-dimethylquinoline, dehydrodemethylethoxyquin (DHMEQ) Pears3,4

N CH3

CH3 Et O 6-Ethoxy-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline, dihydroethoxyquin Pears3,4 CH3 (DHEQ) N H CH3

CH3 Et O 6-Ethoxy-2,4-dimethyl-1,2-dihydroquinoline, Pears3,4 demethylethoxyquin (DMEQ) N CH H 3

Glu: glucuronide, G: glutathione, Et: ethyl group (C H ). 2 2 5 1Burka et al. [21], Berdikova Bohne et al. [26], 3Gupta and Boobis [14], 4JMPR [25].

A and E in blood plasma was observed [40]. This finding molecules can be formed, for example, 2,6-dihydro-2,2,4- suggests that an organism is using EQ instead of natural trimethyl-6-quinolone (QI) or nitroxide radical (6-ethoxy- antioxidants. High efficiency of this antioxidant results not 2,2,4-trimethyl-1,2-dihydroquinolin-N-oxyl) which is also a only from chemical features of EQ itself but also from the strong antioxidant [39, 42]. fact that products of its oxidation also possess antioxidative Products of EQ oxidation were detected by different properties [12, 39, 41]. authors in fish oil and meal [22, 43–45]. According to Studies on EQ antioxidant properties were performed He and Ackman [46] the following oxidization products by Taimr [39] with the use of alkylperoxyls, and it was of EQ dominate in fish meal and fish feed: 2,6-dihydro- 󸀠 shown that the reaction rate of EQ with them is very 2,2,4-trimethyl-6-quinolone (QI) and 1,8 -di(1,2-dihydro-6- high. In the presence of high oxygen concentrations EQ ethoxy-2,2,4-trimethylquinoline) (EQDM). At high storage reacts with alkylperoxyl molecule to form aminyl radical (6- temperature neither QI nor EQDM accumulates; how- ethoxy-2,2,4-trimethyl-1,2-dihydroquinolin-1-yl) which sub- ever, another product of EQ oxidation, 2,4-dimethyl-6- sequently may enter various pathways. In nonoxidizing con- ethoxyquinoline, is stable [41]. As it was pointed out earlier, ditions it can be stabilized both by the loss of methyl group ethoxyquin oxidization products also possess antioxidative and aromatization of heterocycle to form 2,4-dimethyl-1,2- properties. The EQDM and QI show 69% and 80% of EQ dihydroquinoline (dehydrodemethylethoxyquin (DHMEQ)) efficacy, respectively (studies on fish meal) [12]. On the other and through dimerization to form EQ dimer (EQDM) [39, hand, in the studies of Thorisson et al. [45] quinone imine 42]. On the other hand, in an oxidizing medium other (QI) and EQ nitroxide were also powerful antioxidants, while 6 International Journal of Food Science

CH H3C 3 O

CH3 N H CH3

Ethoxyquin 6-Ethoxy-1,2-dihydro-2,2,4-trimethylquinoline

CH 3 CH3 HO H

CH3 CH3 N N H CH3 H CH3

Hydroxyquin Hydroquin 2,2,4-Trimethyl-1,2-dihydroquinolin-6-ol 2,2,4-Trimethyl-1,2-dihydroquinoline

CH H3C H3C H H 3 O

CH3 CH3 N N H3C H3C H H CH3 CH3 6-Ethoxy-2,2,5,7-tetramethyl- 2,2,4,7-Tetramethyl-1,2,3,4- 1,2,3,4-tetrahydroquinoline tetrahydroquinoline

Figure 1: Chemical structure of ethoxyquin (EQ) and of some new compounds synthesized on ethoxyquin backbone with promising antioxidant properties (according to de Koning [12], Dorey et al. [15] and Błaszczyk and Skolimowski [17]).

EQDM,themainproductofEQoxidation,showedlittleorno in the case of lower EQ concentrations (5 𝜇M, 10 𝜇M) with no antioxidant behavior. effect at higher concentration17 [ ]. AntioxidantactivityofEQwasalsodemonstratedin experiments performed both in vivo and in vitro.Antimu- tagenic effect of this antioxidant was observed in mice, rats, 5. Adverse Effects of EQ: In Vivo and and Chinese hamsters treated with cyclophosphamide, an In Vitro Studies agentwidelyusedincancerchemotherapy[47–49]. During cyclophosphamide bioactivation reactive oxygen species are Different phenolic antioxidants may be used in animal feed, formed which can cause damage of genetic material [50, such as BHA (butylated hydroxyanisole), BHT (butylated 51]. EQ reduced the number of chromosome aberrations, hydroxytoluene), and the most efficacious EQ. The levels of micronuclei, and dominant lethal mutations induced by the the antioxidants in finished feed should not be higher than anticancer drug [47–49]. There were also some reports that 150 ppm for EQ and 200 ppm for BHT and BHA (U.S. Food EQ can modify carcinogenic response to different carcino- and Drug Administration permissions). The fact that efficient gens [35, 52, 53].EQgiventoFischer344ratsindiet antioxidants work optimally when they are used at low con- completely prevented the formation of aflatoxin B1-induced centrations is their remarkable characteristic. On the other preneoplastic liver lesions [52, 53]. hand, when antioxidants are used at high concentrations they In in vitro experiments with human lymphocytes, antiox- act as prooxidants. The impact of these compounds depends idantactivityofEQwasobservedinthecometassay(the on their concentration as well as on other factors such method used to detect single- and double-strand DNA as metal-reducing potential, chelating behaviour, solubility, breaks, cross-links, and alkaline labile sites) and in micronu- and pH. The effect of antioxidants on living organisms also cleus test (the method for the detection of micronuclei depends on their bioavailability and stability in tissues [54, induced by clastogens or aneugens). EQ used at the con- 55]. Phenolic antioxidants under favorable conditions may centrations ranging from 1 𝜇Mto10𝜇Mprotectedhuman beconvertedtophenoxylradicalswithprooxidantactivity lymphocytesagainstDNAdamagecausedbyhydrogenper- [55]. It was shown that dissolved EQ may exist partly in the oxide (H2O2,10𝜇M) [17]. This antioxidant also reduced the free radical form which it was also detected in the compound number of micronuclei caused by H2O2 used at concentration itself [56]. Therefore, ethoxyquin nitroxide which is produced of 75 𝜇M. However, the significant reduction was evident only by EQ oxidation similarly as other nitroxide molecules International Journal of Food Science 7

Table 4: Harmful effects of EQ observed after its oral administration Detrimental effects of EQ were also seen when the in different animals or in humans (contact exposure). experiments were performed at the cell metabolism level. Hernandez et al. [64]andReyesetal.[65]analysedtheimpact The harmful effect Animals of EQ on the metabolic pathways of rat renal and hepatic cells, Marmosets1,rats2,dogs2, mice2, Loss of weight 3 as well as on mitochondria and submitochondrial particles rabbits obtained from bovine heart and kidney. They observed 1 1,4 2 2 Marmosets ,rats ,dogs , mice , influence of EQ on energy processes in cells. EQ inhibited Changes in liver 2 5 + + broiler chickens ,tilapia renal Na ,K -ATPaseactivityinvolvediniontransport[64]. Marmosets1,rats2,6,7,dogs2,broiler Changes in kidney 2,8 The authors suggested that EQ interacted with site I of the chickens mitochondrial respiratory chain, and it resulted in inhibition Marmosets1,dogs2, mice2,broiler Changes in alimentary duct 2,8 of oxygen consumption in the mitochondria of kidney and chickens liver cells when glucose was a respiratory substrate. The effect Changes in urinary bladder Rats2 was dose dependent. Anemia Marmosets1 More than 30 years ago when EQ began to be more Changes in mitochondria Rats2 commonly used in animal feed research started to assess its Lethargy Rabbits3 mutagenicity with the use of Ames test which is performed on different Salmonella typhimurium strains. The results were Colored urine, skin, or fur Dogs2,rats2 2,8 equivocal as some results were negative [66–68], but the Increase in mortality Broiler chickens positive effects were also observed69 [ , 70]. It was also Effect on immunity Tilapia5 shownthatEQenhancedthemutagenicactivityofDMBA 󸀠 Condition factor: the final (3,2 -dimethyl-4aminobiphenyl), a compound having car- body weight in relation to Largeyellowcroaker9 cinogenicproperties[70]. Ethoxyquin was reported to both body length of fish enhance and inhibit genetic changes induced by known Allergy (contact exposure) Humans10,11,12 carcinogens; on the other hand it can also lead to cancer 1 McIntosh et al. [58], 2Drewhurst [13], 3Little [10],4Ito et al. [59], 5Yamashita in exposed animals. Manson et al. [53] observed in Fischer et al. [60], 6Neal et al. [61], 7Manson et al. [53], 8Leong and Brown [62], 344 rats that EQ caused severe damage in kidney. Many 9 10 11 12 Wang et al. [63], Rodr´ıguez-Trabado et al. [9], Brandao [5], Savini et hyperplastic and putative preneoplastic tubules were found al. [6]. which suggested that EQ may be exerting a carcinogenic effect. Similar effects were observed earlier by Ito et al.59 [ ]in relation not only to the kidney but also to the urinary bladder. Possible carcinogenicity of EQ is probably connected (e.g., tempol) may also show prooxidative properties [57]. with its prooxidant activity and induction of reactive oxygen Formation of free oxygen species as a result of using too high radicals which cause DNA damage. DNA damage is usually EQ concentrations can cause adverse health effects in animals repaired by cellular repair system, but if it is severe or there fed with EQ containing feed or in people consuming meat are too many lesions, this leads to programmed cell death from farmed animals, for example, different fishes. (apoptosis). Sometimes, however, the programmed cell death The studies on EQ prooxidant activity and toxicity associ- pathway is damaged so when the defense mechanisms fail atedwithitwereperformedboth in vivo and in vitro.Dogsare there is no way to stop a cell from becoming a cancer cell. most susceptible to the harmful effects of EQ, and first reports Some in vitro studies showed both cytotoxic effects of EQ of such effects were received by FDA in 1988. The symptoms leadingtocellapoptosisornecrosisanddamageofgenetic observed by dog owners and veterinarians were liver, kidney, material at DNA or chromosome levels. Cytotoxic effects of thyroid and reproductive dysfunction, teratogenic and car- pure EQ (purity > 97%) were studied in vitro with the use of cinogenic effects, allergic reactions, and a host of skin and humanlymphocytes.TheIC50 value (the concentration caus- hair abnormalities [7]. According to the studies on dogs and ing 50% growth inhibition) for EQ determined after 72-hour laboratory animals it was shown that ethoxyquin had little treatmentofthecellsintheMTTassaywas0.09mM[71]. acute toxicity, except when it is administered parenterally. −1 This antioxidant significantly reduced viability of lympho- Values of LD 50 for EQ are 1700 mg kg bw (rats, oral −1 cytes detected with trypan blue exclusion method after 24- gavage), >2000 mg kg bw (rats, dermal treatment, 24 h), −1 hour treatment at the concentrations of 0.25 and 0.5 mM (cell ∼900 mg kg bw (mice, intraperitoneal administration), and divisions were stimulated by phytohemagglutinin, (PHA)) −1 ∼180 mg kg bw (mice, intravenous administration) [13]. [19] or of 0.05 mM and higher when 1-hour treatment was Despite species differences in the majority of animals treated performed [72]. EQ-induced apoptosis by observed in in with EQ at the concentrations higher than those permitted in vitro cultured human lymphocytes starting from 0.05 mM animal feed, the same characteristic symptoms and patholo- concentration and the detected number of apoptotic cells gies appeared such as weight loss, liver, and kidney damage, depended on the treatment time [71]. Ethoxyquin caused also alterationsofalimentaryduct(Table 4). The concentration DNA damage in the comet assay [72] however, most lesions −1 of 100 ppm (equivalent to 2.5 mg kg bw per day) was could be repaired by cellular DNA repair systems [73]. On the considered to be a minimal-effect level for clinical signs of other hand, the results obtained with the use of chromosome toxicity and liver effects in dogs, the most susceptible animals aberration test showed that unrepaired DNA damage induced [13, 14]. by EQ could lead to permanent changes in genetic material 8 International Journal of Food Science

[16, 74]. Błaszczyk et al. [16]andGilleetal.[74]showed Table 5: Comparison of different activities of ethoxyquin and that this antioxidant induced chromosome aberrations such its derivatives based on the data presented by Błaszczyk and as breaks, dicentrics, atypical translocated chromosomes, or Skolimowski [19, 71, 80–82] and Błaszczyk et al. [79]. chromatid exchanges in human lymphocytes and Chinese Activity from the lowest (left) to the highest Activitity hamster ovary cells. These aberrations are known to have (right) one serious biological consequences [75]. EQ-F < EQ-HCl < EQ-C < EQ-S < EQ-T < EQ-Q Cytotoxicity < EQ-R < EQ-H < EQ EQ-F < EQ-C < EQ-H < EQ-HCl < EQ-Q < Genotoxicity 6. Analogues and Derivatives of EQ EQ-T < EQ-S < EQ-R < EQ Antioxidant EQ-F < EQ-HCl < EQ-R < EQ-T < EQ-S < EQ-C Because of adverse health effects caused by EQ it is reasonable activity < EQ-Q < EQ < EQ-H to search for new antioxidants as effective in scavenging free radicals as EQ which produce no such problems. In the EQ-F: ethoxyquin phosphate; EQ-HCl: ethoxyquin hydrochloride; EQ-C: ethoxyquin L-ascorbate; EQ-S: ethoxyquin salicylate; EQ-T: ethoxyquin salt paper of de Koning [12] nine analogues of EQ prepared to of Trolox C; EQ-Q: ethoxyquin complex with quercetin; EQ-R: ethoxyquin compare their antioxidant efficacy with that of the parent complex with rutin; EQ-H: ethoxyquin 𝑛-hexanoate; EQ: ethoxyquin. chemicalarepresented.Thecompoundshavebeentestedina refinedfishoilandsubsequentlysomeofthemostpromising ones have been also tested in fish meal. It was noted that the use of human lymphocytes in vitro were significantly the results obtained in fish oil were not always the same lower. We believe that this chemical is worth of detailed as in fish meal, for example, hydroxyquin (1,2-dihydro-6- studies to confirm its usefulness as a food preservative. hydroxy-2,2,4-trimethylquinoline; Figure 1) was 3.5 times as Some other EQ derivatives and salts were also studied for effective as EQ in fish oil, while only 3/4 of its efficacy was cytotoxicity, genotoxicity, and antioxidant activity, namely, observed in fish meal. In the case of another compound— the complexes of ethoxyquin with flavonoids (rutin or hydroquin (1,2-dihydro-2,2,4-trimethylquinoline; Figure 1) quercetin), ethoxyquin hydrochloride, ethoxyquin phos- antioxidant efficacy in relation to EQ was 101% in fish oil phate, ethoxyquin L-ascorbate, ethoxyquin n-hexanoate, and52%infishmeal.Despitethelowerefficiencyofthis ethoxyquin salicylate, and ethoxyquin salt of Trolox C [19, compound in fish meal the author stated that hydroquin 79–82]. The biological properties of the compounds were can compete with EQ as an antioxidant of choice [12]. The analysedwiththeuseofMTT,TUNEL,andtrypanbluestain- reason is that preparation of hydroquin based on aniline ing methods (cytotoxicity testing), comet assay (genotoxicity and acetone is more cost-effective than that of EQ whose testing), and micronucleus test (mutagenicity testing). From production requires p-phenetidine (more expensive than among the compounds tested ethoxyquin phosphate (EQ- aniline). Hydroquin was earlier patented as an antioxidant F) was the least toxic (Table 5)—its cytotoxic and genotoxic in animal feeds in 1997 [76]. The 2-year dermal research activities in comparison with those of EQ were reduced with the use of F344/N rats and B6C3F1 mice conducted positively (IC50 = 0.8 mM versus 0.09 mM for EQ) [71]. On under the National Toxicology Program [77] showed that the the other hand, antioxidant activity of EQ-F was observed, compound was not carcinogenic, but the studies performed butitwasthelowestofthetestedcompounds[82]. The studies by Sitarek and Sapota [78] showed its teratogenic properties. showed that all the tested compounds were less toxic to In 2000 Dorey et al. [15] presented the report concerning human lymphocytes than EQ, and the antioxidant activity of the synthesis and biological properties of a new class of four of them (ethoxyquin n-hexanoate, ethoxyquin complex antioxidants based on the EQ backbone. The studies were with quercetin, ethoxyquin L-ascorbate, and ethoxyquin performed to search for new quinolinic derivatives with salicylate) was comparable with that of EQ [79–82]. The radical scavenging activity, potential candidates for central results obtained indicate that their use as antioxidants may nervous system protection. EQ is not suitable for that as it be considered. has been shown to exhibit significant hypothermic effect, probablyasaresultofaninhibitionofelectrontransport 7. Food Safety Aspect in the mitochondrial respiratory chain [65]. Dorey et al. [15] synthesized and studied many 1,2-dihydro and 1,2,3,4- EQ safety has been under consideration for many years. tetrahydroquinolines and then selected for further evalu- The level of this antioxidant in animal feeds should notbe ation a group of antioxidants (5 compounds) with high higher than 150 ppm (U.S. Food and Drug Administration radical scavenging capacities, relatively low toxicity, and permissions). The approved uses of ethoxyquin in animal moderate hypothermia. The compounds belonging to the feeds are addressed in the Code of Federal Regulations (CFR), group of 1,2,3,4-tetrahydroquinolines (e.g., 6-ethoxy-2,2,5,7- Title 21, Parts 573.380 and 573.400, and established tolerances tetramethyl-1,2,3,4-tetrahydroquinoline, characterized with are in Part 172.140. On the one hand, the observed adverse the lowest toxicity and high radical scavenger capacity) health effects (firstly in dogs) could be caused by the fact are structurally similar to 2,2,4,7-tetramethyl-1,2,3,4-tetrahy- that the animals ate a lot of feed containing EQ, but on droquinoline synthesized and tested in our laboratory the other hand, it could also be the result of its excessive (Figure 1)[17]. The latter compound also had promising amounts in the feed. Ethoxyquin is added to animal feed features: its antioxidant activity was comparable to that either directly or indirectly as a component of an ingredient. of EQ, but its cytotoxicity and genotoxicity studied with From time to time FDA reminds industry about labeling and International Journal of Food Science 9 safe use requirements for ethoxyquin, but if it is added at the higher than that of EQ. The authors estimated that consumer ingredient level this is not always indicated. exposuretoEQfromasingleportion(300g)ofskinned- Another important safety issue is the presence of EQ fillets of different species of farmed fish could amount upto oxidation and EQ metabolism products in animal feed or 15% of the ADI. In the light of data concerning the presence in foods prepared from farmed animal meat. de Koning of EQDM in the body of farmed fishesand providing that EQ [12] described main products of EQ oxidation which can be dimer was included in the ADI, the EQ and EQDM intake observed in stored feeds or in fish meal: EQ dimer (EQDM, from a single portion of Atlantic salmon would be close to 󸀠 1,8 -di(1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline) and ADI [24].FarmedfishisprobablythemajorsourceofEQand quinone imine (QI, 2,6-dihydro-2,2,4-trimethyl-6-quin- itsresiduesforEuropeanconsumers(itsuseasafoodadditive olone). Both compounds were shown to be potent antioxi- is forbidden). In our opinion, however, both fish and other dants, but they can also have detrimental effect, especially so farmed animals, for example, chickens, should be controlled because the half-life of the dimer was considerably greater for the presence of not only EQ, but also EQDM, its main than that of EQ [26, 28]. In the recent studies no adverse oxidation product. toxicological effects of EQDM, in terms of kidney and liver function, were observed in in vivo experiments with F344 8. Conclusion rats exposed for 90 days to the compound [37]. On the other hand, Augustyniak et al. [83]showedthatEQDM, Ethoxyquin has been used as an antioxidant in animal feed similarly as EQ, was cytotoxic and genotoxic to human for several decades and despite the search for new compounds lymphocytes. Toxicity of QI has not been studied yet, but the thatcouldbeusedasfreeradicalscavengers,itisstillthemost results obtained by the authors indirectly indicated that the effective antioxidant. The negative health effects in domestic compound could be cytotoxic to human cells. animals fed with EQ containing feed were observed some The levels of the parent compound (EQ) in meat of years ago, but the presence of its approved doses should not farmed animals are usually lower than MRL (Maximum be hazardous. Toxicity and mutagenicity of EQ were observed Residue Level) [20, 84], but EQ oxidation products are in in vivo and in vitro studies showing its potential harmful usually not controlled. It was shown that EQDM and other effects. This makes it very important to label all products and EQ residues can be present in different animal tissues [23, ingredients to which EQ is added and to comply with the 24, 26, 28, 37]. In the studies of Bohne et al. [23]in recommended doses. Additionally, the results of the studies which Atlantic salmons were fed for 12 weeks with the feed on products of EQ oxidation, especially EQDM, detected in containing this antioxidant, four compounds were identified farmed animal tissues indicate that it should be under control in their muscles: parent EQ (6-ethoxy-1,2-dihydro-2,2,4- and some regulations should be introduced. trimethylquinoline), deethylated EQ (6-hydroxy-2,2,4-trim- ethyl-1,2-dihydroquinoline), quinone imine (2,6-dihydro- 󸀠 Conflict of Interests 2,2,4-trimethyl-6-quinolone, QI), and EQ dimer (1,8 -di(1,2- dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQDM). It was The authors declare that there is no conflict of interests. also shown that the concentration of EQ in fish muscle was proportional to the duration of exposure and the level of EQ in the feed [23]. The same linear increase was seen for References EQDM, the main metabolite of EQ, and the sum of EQ and [1] H. 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